An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq.^[1]

A spike-in is designed to bind to a DNA molecule with a matching sequence, known as a control probe.^[2][3][4] This process of specific binding is called hybridization. A known quantity of RNA spike-in is mixed with the experiment sample during preparation.^[2] The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements of the sample RNA.^[2]

History

Nucleic acid hybridization assays have been used for decades to detect specific sequences of DNA or RNA,^[5] with a DNA microarray precursor used as early as 1965.^[6] In such assays, positive control oligonucleotides are necessary to provide a standard for comparison of target sequence concentration, and to check and correct for nonspecific binding; that is, incidental binding of the RNA to non-complementary DNA sequences.^[7] These controls became known as "spike-ins".^[1] With the advent of DNA microarray chips in the 1990s^[8] and the commercialization of high-throughput methods for sequencing and RNA detection assays, manufacturers of hybridization assay "kits" started to provide pre-developed spike-ins.^[1] In the case of gene expression assay microarrays or RNA sequencing (RNA-seq), RNA spike-ins are used.

Manufacturing

RNA spike-ins can be synthesized by any means of creating RNA synthetically, or by using cells to transcribe DNA to RNA in vivo (in cells).^[1] RNA can be produced in vitro (cell free) using RNA polymerase and DNA with the desired sequence.^[1] Large scale biotech manufacturers produce RNA synthetically via high-throughput techniques and provide solutions of RNA spike-ins at predetermined concentration.^[1] Bacteria containing DNA (usually on plasmids) for transcription to spike-ins are also commercially available.^[1] The purified RNA can be stored long-term in a buffered solution at low temperature.^[1]

Applications

DNA microarrays

DNA microarrays are solid surfaces, usually a small chip, to which short DNA polymers of known sequence are covalently bound.^[6] When a sample of unknown RNA is flowed over the array, the RNA base pairs with and binds to complementary DNA.^[6] Bound transcripts can be detected, indicating the presence of RNA with the corresponding sequence.^[6] DNA microarray assays are useful in studies of gene expression, because many of the mRNA transcripts present in a cell can be detected at the same time.^[6] RNA spike-ins of known quantity can provide a baseline signal for comparison with the signal from transcripts of unknown quantity, such that the data can be normalized within an array and between different arrays.^[2]

Sequencing

RNA sequencing (RNA-Seq) is performed by reverse transcribing RNA to complementary DNA (cDNA) and high-throughput sequencing the cDNA.^[9] Such high-throughput methods can be error prone, and known controls are necessary to detect and correct for levels of error.^[9] RNA spike-in controls can provide a measure of sensitivity and specificity of an RNA-Seq experiment.^[9]